Two factors are mainly responsible for the stability of the DNA double helix: base pairing between complementary strands and stacking between adjacent bases. By studying DNA molecules with solitary nicks and gaps we measure temperature and salt dependence of the stacking free energy of the DNA double helix. For the first time, DNA stacking parameters are obtained directly (without extrapolation) for temperatures from below room temperature to close to melting temperature. We also obtain DNA stacking parameters for different salt concentrations ranging from 15 to 100 mM Na+. From stacking parameters of individual contacts, we calculate base-stacking contribution to the stability of A*T- and G*C-containing DNA polymers. We find that temperature and salt dependences of the stacking term fully determine the temperature and the salt dependence of DNA stability parameters. For all temperatures and salt concentrations employed in present study, base-stacking is the main stabilizing factor in the DNA double helix. A*T pairing is always destabilizing and G*C pairing contributes almost no stabilization. Base-stacking interaction dominates not only in the duplex overall stability but also significantly contributes into the dependence of the duplex stability on its sequence. For all temperatures and salt concentrations employed in present study, base-stacking is the main stabilizing factor in the DNA double helix. A•T pairing is always destabilizing and G•C pairing contributes almost no stabilization. Base-stacking interaction dominates not only in the duplex overall stability but also significantly contributes into the dependence of the duplex stability on its sequence. Moreover, single-stranded break (a nick) in the DNA double helix is stabilized by stacking interactions between base pairs flanking the lesion; these interactions are sequence-dependent ( 13 ). In the cell, DNA nicks are substrates for DNA damage-detecting and DNA-repair proteins ( 14 – 17 ). In vitro , DNA nick-based approaches are often used in DNA detection and amplification protocols ( 18 , 19 ). Also, stabilization achieved through coaxial stacking interactions has been put into effect to improve the efficiency of short primer hybridization for standard sequencing protocols ( 20 ) and within the format Experimentally, energetics of base-stacking interactions in nucleic acids has been evaluated by studying the effect of dangling (unpaired) terminal bases on the overall stability of duplexes ( 31 – 35 ) and in the coaxial stacking hybridization experiments where binding of a short oligonucleotide to the single-stranded DNA template is assisted by stacking interaction with the nearby duplex interface ( 36 – 41 ). Both of these approaches rely on thermal denaturation measurements of short duplex molecules. We have recently introduced an entirely new approach for characterization of stacking interactions in the DNA double helix ( 13 ). One conformation is very close to that of the intact double helix where stacking between the base pairs flanking the nick is conserved. We assume that no optimization of stacking interactions—like in case of duplexes with dangling nucleotides ( 52 )—occurs at the site of the nick in DNA. The other conformation corresponds to complete loss of stacking at the nick site thus inducing a kink in DNA. The fast equilibration between stacked/straight and unstacked/bent conformations of the nick directly affects the mobility of DNA molecule during PAGE leading to a differential retardation characteristic to a particular dinucleotide carrying the nick, i.e. KL. Here we measure, for the first time, temperature dependence and salt dependence of base-stacking contribution to the DNA duplex stability. Contributions of A•T and G•C pairing are estimated from the comparison of A•T- and G•C-containing polymer stability parameters with the stacking terms. We find that throughout the temperature range employed, base-stacking interactions stabilize DNA double helix. Temperature dependence of the base-stacking term fully determines the temperature dependence of the DNA stability parameter. Base pairing term is destabilizing in case of A•T and somewhat stabilizing in case of G•C pairs. The stacked-unstacked transition of a nicked DNA doublet involves loss of stacking interactions while hydrogen bonding between the complementary bases is preserved ( Figure 6 ). It also involves removal of structural constraints imposed in the double helix making most of the conformational space of the intact sugar-phosphate backbone accessible. This part of backbone conformational entropy gain is close to the gain per one strand upon duplex melting. The conformation about the glycosidic bond in an open stack, however, is preserved as in duplex form. Note, that charge of the phosphate at the 5′ nt of the nicked stack does not affect salt dependence of measured Δ G KL ST parameters. CONCLUSIONS Separation of the two contributions to thermal stability of the DNA double helix is achieved by studying PAGE of DNA molecules with solitary nicks and gaps. For the first time, the dependence of DNA stacking parameters on ambient conditions (temperature and salt concentration) is determined. Throughout the temperature and salt concentration range of our experiments, base-stacking interactions are always stabilizing for both A•T- and G•C-containing contacts in the DNA double helix. In fact, DNA stability is mainly determined by base-stacking interactions. G•C pairing does not contribute to stabilization of DNA duplex, while A•T pairing is always destabilizing. This finding presents a paradigm shift in the understanding of the interplay of the forces stabilizing DNA double helix. For all temperatures heterogeneity of stacking interactions in A•T- and G•C-containing contacts accounts for at least half of heterogeneity in the stability of A•T- and G•C-polymers; the other half is due to the difference in the energetics of A•T and G•C base pairing.